Over the past few decades, nucleic acid technology has grown to assume an indispensable role in many areas of genetic research. Many useful applications such as genotyping, diagnosis of diseases, analysis of biological samples, and many other diagnostic applications have been spawned from research efforts put into this field. Efforts are currently being focussed on the development of DNA biosensors that employ efficient nucleic acid amplification and detection techniques to achieve better sensitivity and specificity within a short turnaround time.
A fundamental process carried out in a DNA biosensor is the transduction of a nucleic acid recognition event, such as the hybridisation of a probe nucleotide sequence with a single stranded DNA from a sample, into a signal that is detectable by conventional detection methods. Amongst the various types of detection methods known in the art, such as optical (light), electrical (current), electrochemical and frequency detection methods, optical detection methods have become very widely employed.
Optical detection methods typically require the use of fluorescent molecules. Hybridisation of target nucleotide sequences labelled with fluorescent molecules (e.g. ethidium bromide) can be detected by monitoring the increase in fluorescence that accompanies the hybridisation between the probe and the target nucleotide sequence. When employed in conjunction with surface modification techniques, fluorescent labels have enabled high density DNA arrays to be constructed. Massively parallel reactions carried out on high density arrays are able to rapidly elucidate nucleic acid sequences, and thus substantially reduce the amount of time required for analysis.
However, fluorescent labels suffer from several shortcomings. For example, tedious procedures are involved in the attachment of the fluorescent molecule to the target DNA. Moreover, only a few of the fluorescence-based detection techniques have sufficiently high sensitivities for the detection of DNA at sub-nanomolar levels. In order to avoid such problems, electrochemical detection methods have been proposed for carrying out ultrasensitive detection of DNA hybridisation events.
Electrochemical DNA biosensors have been reported since about 1990. A wide variety of methods, such as the use of gold nanoparticles, the direct oxidation of guanine, as well as the use of DNA intercalators, have been developed for facilitating electrochemical detection methods. It was known that unlike fluorescent indicators, the use of DNA intercalators as hybridization indicator does not involve the tedious labelling of the target DNA with the fluorescent indicator, as is commonly employed in conventional DNA detection techniques. Additionally, expensive optical equipment are not needed. The inherent miniaturization of electrochemical biosensors and their compatibility with advanced semiconductor technologies also promise to provide a simple, accurate and inexpensive platform for nucleic acid assays.
Both low- and high-density electrochemical DNA sensor arrays have been successfully fabricated. At present, high-density fluorescent microarrays are substantially cheaper to manufacture and implement than high density electrochemical sensor arrays. However, low-density electrochemical sensor arrays have the following advantages over fluorescent arrays: (i) they provide a cost-effective alternative to expensive optical devices; (ii) when coupled with catalysis, they are ultrasensitive; (iii) they provide rapid, direct, turbid and light absorbing-tolerant detection of hybridisation events; and (iv) they are portable, robust, cheap and require only easy-to-handle electrical components. These advantages render electrochemical biosensors suitable for field tests and home-care use.
Two fundamental issues that need to be addressed in the development of catalytic/enzymatic biosensors are the large background noise accompanying the signal and the sensitivity of the sample assay. Currently, many electrochemical biosensors are still plagued with low signal/noise ratios. Most DNA intercalators not only bind to double-stranded DNA (ds-DNA) but also, to a lesser extent, to single-stranded DNA (ss-DNA) molecules by electrostatic interaction. Several approaches for obtaining improved intercalators have been investigated.
Takenaka et al. synthesized a ferrocene-grafted naphthalene diimide (ND) threading intercalator that was reported to bind to ds-DNA more selectively than usual intercalators (Anal. Chem. 2000, 72 1334-1341). Similarly, Makino et al. disclosed a ferrocene-grafted naphthalene diimide threading intercalator reportedly required an electric potential of less than 450 mV for electrochemical detection (U.S. Pat. No. 6,368,807; US Patent Application No. 20020012917 A1). Steullet et al. disclosed a ND threading intercalator in which a pair of ruthenium complexes are each located at the termini of the side chains of the ND scaffold. Each ruthenium complex is coordinatively bonded to a bipyridine group that is carried on a straight-chained amide side chain (First International Electronic Conference on Synthetic Organic Chemistry—ECSOC-1, E0003, Sep. 1-30, 1997). The effect of DNA scaffolding on intermolecular electron transfer quenching of a photoexcited ruthenium (II) polypyridine naphthalene diimide was investigated in a further study, and it was found that the pendant chromophore interacted weakly with the DNA duplex (Inorg. Chem. 1999, 38, 5526-5534).
Further attempts were made by other groups to enhance the sensitivity and to lower the detection limit of detection methods relying on ND threading intercalators by incorporating chemical and biological amplification mechanisms. (Anal. Chem. 2003, 75, 3267-3269; Anal. Chem. 2002, 74, 4370-4377; Patolsky, F., Katz, E., Willer, I. Angew. Chem., Int. Ed. 2002, 41, 3398-3402). Thorp et al. proposed an electrocatalytic scheme for the direct detection of DNA, using the homogeneous electrocatalysts ruthenium-2,2′-bipyridine or osmium-2,2′-bipyridine complex (Anal. Chem. 2000, 72, 3764-3770; Anal. Chem. 2003, 75, 6586-6592). Others have used DNA-enzyme conjugates as bio-electrocatalysts for the electrochemical transduction of DNA recognition events. Bio-catalytic conjugates that are able to associate with DNA recognition events and stimulate the precipitation of an insoluble product on electrodes were also used as an amplification system for DNA sensing (Anal. Chem. 2004, 76, 1611-1617).
In studies where transitional redox active metal complexes were used as homogenous catalysts, the analytical signal obtained was found to be superimposed onto an intrinsically large and fluctuating background current which obscured the analytical signal indicating the occurrence of hybridisation. The large background current was determined to be the result of the direct oxidation of the catalyst, and the catalytic oxidation of the oligonucleotide capture probes (CP) by the catalyst (Anal. Chem. 2000, 72, 3764-3770; Anal. Chem. 2003, 75, 6586-6592). One solution that has been developed towards eliminating the catalytic oxidation current is to replace the oligonucleotide CP with peptide nucleic acid. However, the problem of direct oxidation of the catalyst was not adequately dealt with. In enzyme-based DNA assays, the background current is known to be directly associated with non-DNA related enzyme uptake, such as non-specific adsorption and electrostatic interaction.
Efforts to bring about the reduction of the background current in catalytic DNA biosensors are currently being made. In one recent study, it was found that background current can be reduced by constructing the DNA biosensor in a bilayer configuration (Analyst, 1995, 120, 2371-2376). As few as 600 copies of target DNA molecules in 1.0 μL droplets were successfully detected with a biosensor having such a bilayer configuration.
Despite the developments that have taken place, there still exists limitations in the present catalytic biosensors for which continuing efforts are needed to improve their performance without incurring prohibitively high manufacturing costs.
Accordingly, it is an object of the present invention to provide compounds which can be used as DNA threading intercalators that have improved detection sensitivity, which are inexpensive to manufacture and simple to use, and thus would enable microarraying techniques to be more widely utilised in biomedical research and healthcare.